Bottom Line:
However, whether Aβ43 is toxic in vivo is currently unclear.In addition, we demonstrate that Aβ43 species are able to trigger the aggregation of the typically soluble and non-toxic Aβ40, leading to synergistic toxic effects on fly lifespan and climbing ability, further suggesting that Aβ43 peptides could act as a nucleating factor in AD brains.Altogether, our study demonstrates high pathogenicity of Aβ43 species in vivo and supports the idea that Aβ43 contributes to the pathological events leading to neurodegeneration in AD.

ABSTRACTThe involvement of Amyloid-β (Aβ) in the pathogenesis of Alzheimer's disease (AD) is well established. However, it is becoming clear that the amyloid load in AD brains consists of a heterogeneous mixture of Aβ peptides, implying that a thorough understanding of their respective role and toxicity is crucial for the development of efficient treatments. Besides the well-studied Aβ40 and Aβ42 species, recent data have raised the possibility that Aβ43 peptides might be instrumental in AD pathogenesis, because they are frequently observed in both dense and diffuse amyloid plaques from human AD brains and are highly amyloidogenic in vitro. However, whether Aβ43 is toxic in vivo is currently unclear. Using Drosophila transgenic models of amyloid pathology, we show that Aβ43 peptides are mainly insoluble and highly toxic in vivo, leading to the progressive loss of photoreceptor neurons, altered locomotion and decreased lifespan when expressed in the adult fly nervous system. In addition, we demonstrate that Aβ43 species are able to trigger the aggregation of the typically soluble and non-toxic Aβ40, leading to synergistic toxic effects on fly lifespan and climbing ability, further suggesting that Aβ43 peptides could act as a nucleating factor in AD brains. Altogether, our study demonstrates high pathogenicity of Aβ43 species in vivo and supports the idea that Aβ43 contributes to the pathological events leading to neurodegeneration in AD.

Fig9: Combining Aβ40 with Aβ43 enhanced Aβ40 immunoreactivity in fly compound eyes. Cryosections from heads of Aβ40+Aβ40- and Aβ40+Aβ43-expressing fly lines and of the GMR-Gal4 driver control line were probed with an Aβ40-specific antibody (green) and counterstained with DAPI (blue) and the number of Aβ40-positive events per eye section was quantified (****p < 0.0001, Student’s t test). Scale bar 50 µm

Mentions:
To verify this assumption and to further assess the potential seeding properties of Aβ43, we performed fractionation of Aβ according to its solubility in both Aβ40+Aβ43 and Aβ40+Aβ40 combined lines. Using the 6E10 pan-Aβ antibody (Fig. 7a), we observed a dramatic decrease of Aβ solubility in the combined Aβ40+Aβ43 condition as compared to the Aβ40+Aβ40 line, with insoluble Aβ species representing 57.89 ± 0.79 % of the total Aβ pool in the former line (****p < 0.0001, vs. Aβ40+Aβ40, Student’s t test, Fig. 7a, b). To determine whether this insoluble Aβ pool was holding Aβ40 species, which are intrinsically mainly soluble, we analysed the soluble and insoluble protein fractions using an antibody specifically targeting Aβ40 species (Fig. 8a). This revealed a progressive shift of Aβ40 species towards the insoluble fraction in the Aβ40+Aβ43 line as compared to the Aβ40+Aβ40 condition, the former having 33.37 ± 1.04 % of its Aβ40 species located in the SDS-insoluble fraction after 5 days of induction (vs. 8.64 ± 0.50 % for the Aβ40+Aβ40 line, ***p < 0.001, Student’s t test, Fig. 8b), while this proportion reached 87.11 ± 2.09 % after 14 days of induction (vs. 30.55 ± 2.49 % for the Aβ40+Aβ40 line, ****p < 0.0001, Student’s t test, Fig. 8c), implying that Aβ43 was a potent trigger of Aβ40 aggregation in vivo. To strengthen this observation, we performed immunofluorescence experiments on cryosections from Drosophila heads following Aβ over-expression in the compound eye, using an antibody specific to Aβ40. While the signal appeared predominantly diffuse in the Aβ40+Aβ40 expressing line, we observed a marked accumulation of Aβ40 species in the eye of the Aβ40+Aβ43 transgenic line (****p < 0.0001, Aβ40+Aβ40 vs. Aβ40+Aβ43, Student’s t test, Fig. 9), which is in line with the biochemical shift of Aβ40 species towards the insoluble protein fraction that we observed in the latter combination (Fig. 8). Altogether, our data indicate that Aβ43 species influence Aβ40 properties leading to its decreased solubility, which subsequently results in a significant enhancement of toxicity in vivo.Fig. 7

Fig9: Combining Aβ40 with Aβ43 enhanced Aβ40 immunoreactivity in fly compound eyes. Cryosections from heads of Aβ40+Aβ40- and Aβ40+Aβ43-expressing fly lines and of the GMR-Gal4 driver control line were probed with an Aβ40-specific antibody (green) and counterstained with DAPI (blue) and the number of Aβ40-positive events per eye section was quantified (****p < 0.0001, Student’s t test). Scale bar 50 µm

Mentions:
To verify this assumption and to further assess the potential seeding properties of Aβ43, we performed fractionation of Aβ according to its solubility in both Aβ40+Aβ43 and Aβ40+Aβ40 combined lines. Using the 6E10 pan-Aβ antibody (Fig. 7a), we observed a dramatic decrease of Aβ solubility in the combined Aβ40+Aβ43 condition as compared to the Aβ40+Aβ40 line, with insoluble Aβ species representing 57.89 ± 0.79 % of the total Aβ pool in the former line (****p < 0.0001, vs. Aβ40+Aβ40, Student’s t test, Fig. 7a, b). To determine whether this insoluble Aβ pool was holding Aβ40 species, which are intrinsically mainly soluble, we analysed the soluble and insoluble protein fractions using an antibody specifically targeting Aβ40 species (Fig. 8a). This revealed a progressive shift of Aβ40 species towards the insoluble fraction in the Aβ40+Aβ43 line as compared to the Aβ40+Aβ40 condition, the former having 33.37 ± 1.04 % of its Aβ40 species located in the SDS-insoluble fraction after 5 days of induction (vs. 8.64 ± 0.50 % for the Aβ40+Aβ40 line, ***p < 0.001, Student’s t test, Fig. 8b), while this proportion reached 87.11 ± 2.09 % after 14 days of induction (vs. 30.55 ± 2.49 % for the Aβ40+Aβ40 line, ****p < 0.0001, Student’s t test, Fig. 8c), implying that Aβ43 was a potent trigger of Aβ40 aggregation in vivo. To strengthen this observation, we performed immunofluorescence experiments on cryosections from Drosophila heads following Aβ over-expression in the compound eye, using an antibody specific to Aβ40. While the signal appeared predominantly diffuse in the Aβ40+Aβ40 expressing line, we observed a marked accumulation of Aβ40 species in the eye of the Aβ40+Aβ43 transgenic line (****p < 0.0001, Aβ40+Aβ40 vs. Aβ40+Aβ43, Student’s t test, Fig. 9), which is in line with the biochemical shift of Aβ40 species towards the insoluble protein fraction that we observed in the latter combination (Fig. 8). Altogether, our data indicate that Aβ43 species influence Aβ40 properties leading to its decreased solubility, which subsequently results in a significant enhancement of toxicity in vivo.Fig. 7

Bottom Line:
However, whether Aβ43 is toxic in vivo is currently unclear.In addition, we demonstrate that Aβ43 species are able to trigger the aggregation of the typically soluble and non-toxic Aβ40, leading to synergistic toxic effects on fly lifespan and climbing ability, further suggesting that Aβ43 peptides could act as a nucleating factor in AD brains.Altogether, our study demonstrates high pathogenicity of Aβ43 species in vivo and supports the idea that Aβ43 contributes to the pathological events leading to neurodegeneration in AD.

ABSTRACTThe involvement of Amyloid-β (Aβ) in the pathogenesis of Alzheimer's disease (AD) is well established. However, it is becoming clear that the amyloid load in AD brains consists of a heterogeneous mixture of Aβ peptides, implying that a thorough understanding of their respective role and toxicity is crucial for the development of efficient treatments. Besides the well-studied Aβ40 and Aβ42 species, recent data have raised the possibility that Aβ43 peptides might be instrumental in AD pathogenesis, because they are frequently observed in both dense and diffuse amyloid plaques from human AD brains and are highly amyloidogenic in vitro. However, whether Aβ43 is toxic in vivo is currently unclear. Using Drosophila transgenic models of amyloid pathology, we show that Aβ43 peptides are mainly insoluble and highly toxic in vivo, leading to the progressive loss of photoreceptor neurons, altered locomotion and decreased lifespan when expressed in the adult fly nervous system. In addition, we demonstrate that Aβ43 species are able to trigger the aggregation of the typically soluble and non-toxic Aβ40, leading to synergistic toxic effects on fly lifespan and climbing ability, further suggesting that Aβ43 peptides could act as a nucleating factor in AD brains. Altogether, our study demonstrates high pathogenicity of Aβ43 species in vivo and supports the idea that Aβ43 contributes to the pathological events leading to neurodegeneration in AD.